Quantifying Changes in the Cellular Thiol-Disulfide Status during Differentiation of B Cells into Antibody-Secreting Plasma Cells

Figure 1

Flow chart of the experimental approach for global quantification of cellular redox species. To avoid contribution of protein disulfides from serum, cells were suspended and washed in phosphate buffered saline prior to addition of trichloroacetic acid (TCA) to 10%. After centrifugation, soluble GSH and GSSG were quantified from the supernatant while protein sulfhydryls were quantified from the pellet. The TCA pellet was divided into four samples (A-D). Sample A was directly incubated with 4,4′-dithiodipyridine (4-DPS) to quantify PSH. To quantify PS_ox, free thiols in sample B were first alkylated with N-ethylmaleimide (NEM). Disulfides then were reduced using sodium borohydride (BH) followed by thiol quantification with 4-DPS. The main advantage of this strategy is that thiol alkylation, disulfide reduction, and thiol quantification can be performed in the same test tube as excess NEM is inactivated by BH, whereas excess BH is easily removed by the addition of acid. As a control, Total PS was measured experimentally in sample C by directly reducing disulfides with BH followed by thiol quantification with 4-DPS. Finally, PSSG in sample D was quantified by reduction of disulfides with tris(hydroxypropyl)phosphine (THP) and fluorescent labeling of thiols with 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F). Selective quantification of GS-SBD derivative was performed using HPLC as described [3]. In addition to quantification of redox species, the total protein content in each pellet was quantified and used as a common denominator to compare the individual samples.